Hyaluronic Acid-Based Multilayer Films Regulate Hypoxic Multicellular

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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

Hyaluronic Acid-Based Multilayer Films Regulate Hypoxic Multicellular Aggregation of Pancreatic Cancer Cells with Distinct Cancer Stem-Cell-like Properties I-Chi Lee,*,†,‡ Yu-Chieh Wu,† and Wei-Shan Hung† †

Graduate Institute of Biomedical Engineering, Chang-Gung University, Taoyuan 33302, Taiwan Neurosurgery Department, Chang Gung Memorial Hospital, Linkou 33305, Taiwan

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ABSTRACT: In vitro spherical cancer models have been widely used in cancer stem cell (CSC) research, and the ability of CSCs to form multicellular colonies is recognized as a morphological marker. However, although several spherical/colony models share a common three-dimensional (3D) conformation, each model displays its own intrinsic properties. Thus, the CSC phenotypes with distinct multicellular aggregate morphologies must be defined and clarified. Here, a novel 3D model was designed to regulate the type of pancreatic CSC colonies that form using niche mimetic hyaluronic acid (HA)-based multilayer nanofilms and hypoxia. The multicellular aggregate morphology, CSC phenotypes, CSC-related marker expression, cell cycle, invasion, and drug resistance were determined. On the basis of the results of a cell morphology analysis, colonies formed on multilayer nanofilms in response to both normoxia and hypoxia, but round and island-type colonies, were investigated. Immunostaining results revealed a significantly higher expression of stem cell markers, such as OCT4, CXCR4, and CD44v6, in colonies that formed on multilayer nanofilms. These colonies also expressed higher levels of E-cadherin, hypoxia-inducible factor-1α, and vimentin, particularly the round-type colonies that formed on HA-based multilayer nanofilms, [poly(allylamine) (PAH)/HA]3, indicating that these colonies exhibit hybrid and metastable epithelial/mesenchymal phenotypes. Moreover, the cell cycle and invasion tests revealed that most of the cells in colonies growing on multilayer nanofilms showed a quiescent, slow cycling phenotype but displayed higher invasion after induction. Furthermore, a hypoxic environment strongly influences the drug resistance. This study describes a useful tool to investigate the diverse phenotypes of pancreatic CSC colonies and to study their regulatory factors that may benefit CSC research. KEYWORDS: pancreatic cancer stem cells, hyaluronic acid-based multilayer nanofilms, hypoxia, colony morphology, stem cell markers, invasion properties

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INTRODUCTION On the basis of the accumulating evidence, tumors are composed of a heterogeneous cell population in which a small subset of cancer stem cells (CSCs) or cancer-initiating cells are endowed with characteristics similar to stem cells and are responsible for tumor initiation, progression, and metastasis.1,2 The CSC hypothesis provides an attractive model of carcinogenesis and helps to explain the possible mechanism of drug resistance and tumor recurrence. The CSC model has provided researchers with a new perspective on the origin of cancer and inspires new strategies for therapy. Pancreatic adenocarcinoma is the fourth leading cause of cancer-related © 2018 American Chemical Society

mortality in western countries and has become one of the most frustrating diseases because of its extremely poor prognosis.3 The high mortality and the 5-year survival rate of 1−3% indicate that the efficacy of treatments for pancreatic cancers has not improved significantly over the past decade, and few therapeutic options are currently available. Gemcitabine has been used as the first-line agent for the treatment of pancreatic cancer; however, the drug resistance develops after a few cycles Received: August 15, 2018 Accepted: October 23, 2018 Published: November 5, 2018 38769

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

ACS Applied Materials & Interfaces

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of administration, affecting the treatment efficacy.4 The existence of pancreatic CSCs has also been reported in several studies which may be the reason for drug resistance.5−7 Therefore, new therapeutic options for pancreatic cancer are urgently needed, and the unique biological characteristics of pancreatic cancer and the specific mechanisms underlying its obstinate malignancy must be determined. Three-dimensional (3D) cell culture systems have been exploited to mimic in vivo environments and have also been widely used in CSC studies as an intermediate model between 2D in vitro cell culture models and in vivo tumor analyses.8 The ability of cells to form multicellular aggregates, such as tumorspheres, colonies, and tumor organoids, has been used as a morphological marker to identify and select malignant cancer cells or CSCs.9−11 However, the properties and classification of different types of cellular aggregates and spheroids formed by cancer cells are less reported, and only Eguchi et al., has tried to use several cell lines to form cell aggregates and discussed about the CSC properties of these cellular aggregation.9 In addition, although several spherical models share a common 3D conformation, each displays its own intrinsic properties.9,12 Thus, studies aiming to determine and clarify the relevance of distinct spherical morphologies and CSC phenotypes are important. In addition, oxygen tension is tightly regulated in normal physiology and is an important signal in development, as low oxygen tension is associated with the maintenance of an undifferentiated cell state. The hypoxic niche plays an essential role in regulating the stemlike biological properties of CSCs.13 Hypoxia promotes the self-renewal of embryonic stem cells and CSCs and prevents the differentiation of stem cells in vitro.14,15 Hypoxia triggers an alteration in cell morphology from an epithelial to a mesenchymal phenotype, which is more pronounced in CSCs.16 Traditional culture methods for stem cells are performed by employing environmental oxygen levels of 18%, whereas most pancreatic adenocarcinomas grow in a pronounced hypoxic tumor microenvironment.17 However, the mechanism by which hypoxia regulates pancreatic CSC properties remains unclear. Niche surrounding the extracellular matrix (ECM) such as hyaluronic acid (HA) plays an essential role in anchoring CSCs to their microenvironment. On the basis of accumulating evidence, HA-CD44 interactions promote tumor-cell-specific behaviors, such as tumor growth and metastasis.18−20 Moreover, previous study also revealed that the CD44 cleavage regulates the cell−matrix interaction and cell migration which may affect pathological processes of tumor.21,22 Therefore, we used HA-based multilayer films to select hepatocarcinoma colonies and to identify their CSC properties in our previous study.23 Here, two different layers of HA-based multilayer nanofilms were designed to form distinct colony types, and the colony morphologies and CSC properties were investigated and compared. Although a relationship between colony formation and CSC properties has recently been proposed, the mechanisms regulating the morphology of aggregates and CSC properties have not been completely elucidated. In the present study, a novel system of HA-based multilayer nanofilms combined with hypoxic niches was established to mimic the pancreatic CSC microenvironment. We investigated CSC phenotypes in different cell aggregates and classified the regulatory factors expressed by diverse CSC phenotypes.

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MATERIALS AND METHODS

Substrate Preparation. A poly(allylamine) (PAH) solution (10 wt % in H2O, Mw ≈ 65 000) and HA sodium salt (Mw ≈ 300 000) were purchased from Aldrich and CarboMer, Inc., USA, respectively. PAH and HA were prepared by direct dissolution in 150 mM Tris buffer (pH 7.4) containing 10 mM NaCl to a final concentration of 3 mg/mL. The PAH solution was sterilized with a 0.22 μm filter (PALL), whereas the HA solution was sterilized under UV light overnight. Glass coverslips were washed with boiling 0.01 M SDS with 0.12 M hydrochloride for 15 min, followed by ddH2O rinses. The coverslips were immersed in 75% ethanol and sterilized under UV light overnight prior to coating using the procedure described in a previous study, with some modifications.23 The PAH solution was first added to coverslips and incubated for 15 min. Normal trisbuffered saline was used to wash coverslips three times to remove the unadsorbed PAH solution. HA solutions were then added and incubated for 15 min, and coverslips were washed as described above. (PAH/HA)n multilayer films were prepared on the coverslips, where n represents the number of absorbed layer pairs. PBS was used to rinse the multilayer films before cells were seeded. Cell Culture and Morphology Observations. The human pancreatic cancer cell line HPAC was a gift obtained from Dr. ChiaNing Shen, Genomics Research Center in Academia Sinica, Taiwan. HPAC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 5% fetal bovine serum (FBS; BioIndustry), 1× insulin-transferrin-selenium (Gibco), 10 ng/ml epidermal growth factor (Peprotech), and a 1% antibiotic antimycotic solution (Gibco) in a humidified 5% CO2 atmosphere at 37 °C. Approximately 1 × 104 HPAC cells were seeded on 24-well plates and incubated on different substrates, including tissue culture polystyrene (TCPS), (PAH/HA)1, and (PAH/HA)3 multilayer films, and individually cultured under two conditions: normoxia, CO2: 5%, O2: 20%, and N2: 75%, and hypoxia, CO2: 5%, O2: 1%, and N2: 94%. The morphology of cells cultured on different substrates was observed using an inverted fluorescence microscope (Leica). Cell Viability and Cytotoxicity Assays. Cells were treated with 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma), MTT, to evaluate the viability at each time point after drug treatment. After 3 and 7 days of culture, 200 or 10 μL of the MTT solution was added to cells in each well of 24-well or 96-well plates, respectively, and incubated for 3 h at 37 °C. Then, the medium was discarded and the resulting MTT formazan crystals were dissolved in 200 or 10 μL of dimethyl sulfoxide (Sigma), respectively. The absorbance of the solutions was then measured at 570 nm using an ELISA reader (BioTek). A cytotoxicity detection kit [lactate dehydrogenase (LDH), Roche] was used to determine the cytotoxicity at each time point. After 3 and 7 days of culture, 100 μL of the assay solution and 100 μL of the culture medium were mixed in 96-well plates and then incubated for 30 min at 37 °C. The absorbance of the solution was then measured at 630 and 490 nm using an ELISA reader (BioTek Instruments, USA). Live and Dead Cell Assay. The viability of HPAC cells cultured on TCPS and different substrates was estimated using the fluorescence-based live and dead assay (LIVE/DEAD kit, Life Technology, USA). After 3 and 7 days of culture, 100 μL of the dye was mixed with 100 μL of the retained medium, added to each well and incubated at 37 °C for 15 min. After the incubation, images of live (green) and dead (red) cells were captured using a confocal microscope (LSM 510 META, Zeiss, Germany). Cell Cycle. Cell cycle analysis was performed using HPAC cells cultured on TCPS and different substrates after 3 and 7 days of incubation. Cells were initially dissociated to a single cell suspension, followed by one wash with ice-cold PBS. Then, cells were fixed with 75% ethanol in PBS for at least 1 h, and the fixative was then removed by centrifugation (2000 rpm for 5 min). A propidium iodide (PI) solution containing 100 μL of a 5 mg/mL PI solution (P4170, Sigma, USA), 100 μL of 10 mg/mL RNase A (EN0531, Invitrogen, USA), and 100 μL of Triton-X100 (T8787, Sigma, USA) in 9700 μL of PBS 38770

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

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Scheme 1. Schematic Illustrating (Not to Scale) the Layer-By-Layer HA-Based Multilayer Nanofilm Formation, HPAC Cell Seeding in Normoxic and Hypoxic Niches, and Different Types of CSC Colonies That Formed

Figure 1. (A) Observations of the morphology of HPAC cells seeded on TCPS, (PAH/HA)1, and (PAH/HA)3 after 3 and 7 days of culture under normoxic and hypoxic conditions. Scale bar = 100 μm. (B) Calculation of spheroid diameters on (PAH/HA)1 and (PAH/HA)3 films after 3 and 7 days of culture under normoxic and hypoxic conditions. The diameters of at least 50 spheroids were estimated for each condition. (C) Calculation of the number of spheroids growing on (PAH/HA)1 and (PAH/HA)3 films after 3 and 7 days of culture under normoxic and hypoxic conditions. At least three whole-field views of 24-well plates were used to count the number of spheroids for each condition. (****, &&&& p < 0.0001). was applied to stain the cells for at least 20 min, and the cell cycle status was determined by flow cytometry (BD Biosciences, USA). Immunostaining. For immunocytochemical characterization, cells were first fixed with ice-cold 4% paraformaldehyde in PBS for 20 min and then washed three times with ice-cold PBS, followed by incubation with 0.1% Triton X-100 for 5 min and three PBS washes. The cells were then incubated overnight at 4 °C with 10% BSA containing the following primary antibodies: Ki-67 (1:200 dilution, Millipore), OCT4 (1:500 dilution, Millipore), CXCR4 (1:500 dilution, Millipore), CD44v6 (1:500 dilution, Millipore), hypoxiainducible factor-1α (HIF1-α; 1:500, Cell Signaling Technology), Ecadherin (1:500 dilution, Sigma), and vimentin (1:500 dilution, Millipore). After discarding the primary antibodies, PBS was used to rinse the cells. Then, the cells were incubated with the following

secondary antibodies or dyes in PBS at room temperature for 1.5 h: FITC-conjugated goat anti-rabbit IgG (1:250 dilution, Millipore), FITC-conjugated goat anti-rat IgG (1:250 dilution, Sigma), FITCconjugated goat anti-Mouse IgG (1:250 dilution, Millipore), rhodamine-conjugated goat anti-mouse IgG (1:250 dilution, Millipore), and Hoechst 33342 (1:5000 dilution, Thermo Fisher). Immunostained cells were visualized using a confocal microscope (LSM 510 META, Zeiss, Germany). The antibodies used in this study were tested and characterized in preliminary studies. Quantification of the marker expression was presented as the area of marker expression over total nuclear expression in colony. Invasion Assay. The invasion assay was performed using transwell cell culture chambers (8 μm pore size, Greiner Bio-One) and an artificial ECM (Matrigel, Corning). Matrigel was prepared in serum38771

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

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ACS Applied Materials & Interfaces free DMEM/F12 on ice at a concentration of 250 μg/mL. One hundred microliters of Matrigel solution were used to coat the transwell chambers, followed by incubation at 37 °C for 2 h. Five hundred microliters of serum-free DMEM/F12 containing 5 × 104 cells were placed in the invasion chambers, and 750 μL of DMEM/ F12 with 20% FBS were added to the bottom chamber as a chemoattractant for the cells. For the induction groups, 750 μL of DMEM/F12 with 20% FBS and 5 ng/mL TGF-β1 were added to the bottom chamber as a chemoattractant for the cells. The cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. After 72 h of culture, the cells were fixed with 3.7% formaldehyde for 2 min and permeabilized with 100% methanol for 20 min. Finally, the cells were stained with crystal violet for 15 min at room temperature, and the cells on the upper membrane in the chambers were removed using a cotton swab. The migrated cells were observed under an inverted microscope (Eclipse-TS100, Nikon, USA), and the areas of crystal violet staining were measured using ImageJ software. Total well and several areas picked up were quantified. The percentage of invaded cells was then determined using the following formula: % invasion = ((total area of cells invading through the Corning Matrigel Matrix‐coated permeable support membrane) /(total area of cells migrating through the uncoated permeable support membrane)) × 100

Drug Resistance Assay: Gemcitabine. Approximately 5 × 103/ well HPAC cells were seeded on 96-well plates on different substrates, including TCPS and glass-based (PAH/HA)n multilayer films and cultured under two conditions, namely, normoxia (CO2: 5%, O2: 20%, and N2:75%) and hypoxia (CO2: 5%, O2: 1%, and N2: 94%), to determine the drug sensitivity of cells isolated from PAH/HA films. After 3 and 7 days of culture, gemcitabine solutions (0, 10, 20, 50, or 100 μmol/L) were added to the wells and incubated for 72 h. Treated cells were incubated with 10 μL of the MTT solution for 3 h at 37 °C. One hundred microliters of dimethyl sulfoxide were added to dissolve the MTT formazan crystals, and the absorbance of the solution was then measured at 570 nm using an ELISA reader (Synergy HT, BioTek Instruments, USA). Statistical Analysis. All data are presented as the mean values ± errors of four independent specimens. Student’s t-test was used to evaluate the statistical significance of differences, and the results are indicated as follows: *, #, or & p < 0.05; **, ##, or && p < 0.01; ***, ### , or &&& p < 0.001; and ****, ####, or &&&& p < 0.0001.

Figure 2. (A) Viability of HPAC cells seeded on TCPS and HA-based multilayer films after 3 and 7 days of culture under normoxic and hypoxic conditions (&&& p < 0.001; ****, ####, &&&& p < 0.0001). (B) LDH release from HPAC cells seeded on TCPS and PEM films and cultured under normoxic and hypoxic conditions for 3 and 7 days.

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Table 1. Percentage of Proliferating Cells Cultured on Different Substrates, TCPS, (PAH/HA)1, and (PAH/HA)3, under Normoxic and Hypoxic Conditions for 3 and 7 Days

RESULTS AND DISCUSSION An illustration of the layer-by-layer adsorption of PAH/HA multilayer nanofilms and the culture conditions is shown in Scheme 1. Cells incubated on (PAH/HA)1 and (PAH/HA)3 substrates in hypoxic and normoxic environments were selected to clarify the effects on colony morphology and CSC phenotypes. The PAH polycation was first coated on the coverslips followed by the polyanion solution, HA. HPAC cells were then sequentially seeded on different substrates after PAH/HA multilayer nanofilms were fabricated. Classification of Multicellular Colony Formation. Approximately 1 × 104 cells/well were seeded on 24-well culture plates, and the cell morphologies and colonies that formed on different substrates under various culture conditions are presented in Figure 1A. HPAC cells adhered to TCPScoated culture wells after 3 days of culture. In contrast, colony formation on (PAH/HA)1 and (PAH/HA)3 multilayer nanofilms was investigated under both normoxic and hypoxic conditions. However, the morphologies of multicellular colonies that formed on (PAH/HA)1 were distinct from those of colonies that formed on (PAH/HA)3. As shown in Figure 1A, the colonies that formed on (PAH/HA)3 exhibited

cell seeding number: 1 × 104 cells/well day day day day day day day day day day day day

3 3 3 3 3 3 7 7 7 7 7 7

normaxia TCPS normaxia (PAH/HA)1 normaxia (PAH/HA)3 hypoxia TCPS hypoxia (PAH/HA)1 hypoxia (PAH/HA)3 normaxia TCPS normaxia (PAH/HA)1 normaxia (PAH/HA)3 hypoxia TCPS hypoxia (PAH/HA)1 hypoxia (PAH/HA)3

proliferation ratio (% of seeding number) 1285 369.5 256 1349.5 353 255 3932.5 2142.7 852.5 1275 475 55

± ± ± ± ± ± ± ± ± ± ± ±

60.7 25.6 5 22.6 12.2 25.5 142.7 270.7 43.3 40.8 82.9 35.1

a typical colony/spheroid morphology with a clear bundle and circular shape. However, the morphology of colonies grown on (PAH/HA)1 was heterogeneous, and not all of the colonies were round; many cell aggregates exhibited the island-type morphology but still displayed a clear bundle. Eguchi et al., 38772

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Figure 3. (A) Live (calcein AM, green) and dead (ethidium homodimer-1 Red) and (B) Ki 67 cell staining of HPAC cells seeded on TCPS and HA-based multilayer films after 3 and 7 days of culture under normoxic and hypoxic conditions. (C,D) Cell cycle analyses of HPAC cells seeded on TCPS- and HA-based multilayer films after 3 and 7 days of culture under normoxic and hypoxic conditions, respectively. (E) Percentages of cells in the G0/G1 phase.

cultured longer, indicating that cell aggregates fused each other and get larger which is consistent with previous literature.24 In addition, the average sizes of the colonies were smaller in the hypoxic culture system than in the normoxic system. The internal microenvironment of the colony and the hypoxia culture system may regulate the cancer cell phenotype and result in CSC enrichment. Cell Viability and Cell Cycle of HPAC Cells Cultured on HA-Based Multilayer Films. The MTT assay was used to evaluate the viability of HPAC cell cultured under different conditions. The number of cells in different groups was fitted to a standard curve according to the optical density (OD) value. Figure 2A shows the number of HPAC cells cultured on TCPS, (PAH/HA)1 and (PAH/HA)3 nanofilms under normoxic and hypoxic conditions. In addition, the quantification of the percentage of seeded cells cultured under different conditions that remained after 3 and 7 days of culture is summarized in Table 1. Fewer cells cultured on (PAH/HA)1 and (PAH/HA)3 films survived after 3 days of culture compared with the surviving number of cells cultured on TCPS, which indirectly suggests a CSC selection and regulatory effect. After 7 days of incubation, greater numbers of cells cultured on TCPS and multilayer nanofilms were

have demonstrated several multicellular aggregation types by using 67 cell lines on NanoCulture Plates and determined their CSC-like properties.9 In comparison with the morphologies defined, it is considered that cells cultured on (PAH/HA)3 displayed morphologies similar to spheroids, and cells cultured on (PAH/HA)1 displayed the morphologies similar to grapelike aggregation. In addition, Figure 1B,C presents the average sizes and colony numbers, respectively, and only round-type colonies with clear bundles were counted. The colony size increased significantly after 7 days of incubation on multilayer nanofilms under all culture conditions compared with the size after 3 days of incubation. In particular, the size of island-type colonies cultured on (PAH/HA)1 increased after 7 days of incubation obviously, the increased degree was larger than round-type colonies. It is considered that the proliferation rates of cells in island-type colonies are higher than those of the typical round-type colonies, and the colonies attend to fuse. Besides, cell migration from the island-type colonies on (PAH/HA)1 also enlarged the colonies’ area and affect the stem-cell-like properties which is consistent with previous studies.24 In contrast, the colony number decreased after 7 days of culture in all groups, as shown in Figure 1C. It is suggested that the spheroid number was reduced when 38773

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

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ACS Applied Materials & Interfaces

Figure 4. Images of immunofluorescence staining in HPAC cells cultured on HA-based multilayer films and the TCPS substrate under normoxic (A) and hypoxic (B) conditions for 7 days. Images in the first to fourth rows present staining for the OCT4, CXCR4, and CD44v6 proteins, respectively (scale bar = 50 μm). (C) Quantification of the fluorescence intensity of OCT4, CXCR4, and CD44v6 staining in cells cultured on different substrates for 7 days (*, p < 0.05; **, p < 0.01, ***, p < 0.001, and ****, p < 0.0001).

higher red fluorescence. Additionally, it is shown that the specific long, island morphology of cells grows on (PAH/HA)1 under hypoxic conditions after long-term culture. We also examined cell proliferation by staining with KI-67 to identify the proliferating part of the colonies. As shown in Figure 3B, cell proliferation is homogeneously distributed throughout island-type colonies. In contrast, in typical round-type colonies, cell division mostly occurs in the peripheral rim which is consistent with live and dead results. This structure is similar to that of a real tumor, which may represent a different tumor model. Spheroids commonly exhibit a concentrically layered structure consisting of a necrotic core surrounded by a viable layer of quiescent cells and an outer rim of proliferating cells.27 As shown in the study by Zhang et al., quiescent cells have a minimal metabolic activity but become active after exposure to nutrients.28 Furthermore, colonies were reseeded on the TCPS substrate, and the cell cycle was examined to identify the cycling status of these colonies. Cells in colonies adhered and migrated from the colonies when these colonies were transferred to the TCPS substrate, indicating that the cells were alive (Figure S1). In particular, cells cultured on (PAH/ HA)3 under hypoxic conditions showed a rapid proliferation rate after reseeding on TCPS and culture in a normoxic environment, as shown in Figure S1B. Furthermore, the cell cycle assay was used to determine the percentage of cells in

observed in the normoxia system, suggesting that cell proliferation occurred. In contrast, the OD values in hypoxia culture systems exhibited a substantial decrease compared with that of cells cultured on TCPS under normoxic conditions. Thus, a hypoxic environment may inhibit cell viability. The activity of LDH released from the cytosol of damaged cells was used to determine cytotoxicity and is also related to metabolism. Figure 2B shows the results of the LDH assay using cells cultured under different conditions for 3 and 7 days. Significant cytotoxicity of these multilayer nanofilms was not observed. In contrast, significantly higher LDH levels were found from the cells cultured on TCPS in a hypoxic environment for 7 days than that from the other groups. Lactate mediates cancer-cell-intrinsic effects on metabolism, and tumor cells metabolize lactate as an energy source, particularly the cells growing in a hypoxic environment.25 Previous study has also revealed that pyruvate was converted to lactate in the hypoxia environment, and HIF-1α can promote the cancer cell viability and also affect metastasis.26 Exposure to hypoxia may reprogram the metabolism of HPAC cells. According to the results of the live and dead cell assay shown in Figure 3A, red fluorescence was obviously detected in both types of colonies cultured on (PAH/HA)1 and (PAH/HA)3 under normoxic conditions. In particular, the colony structure of cells growing on (PAH/HA)3 was very dense and displayed 38774

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

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Figure 5. Immunofluorescence staining in HPAC cells cultured on HA-based multilayer films and the TCPS substrate under normoxic (A) and hypoxic (B) conditions for 7 days. Images in the first to fourth rows present staining for the HIF-1α, E-cadherin, and vimentin proteins, as well as merged photographs, respectively (scale bar = 50 μm). (C) Quantification of the fluorescence intensity of HIF-1α, E-cadherin, and vimentin in cells cultured on different substrates for 7 days.

under normoxic and hypoxic conditions is also shown in Figure 4B. As shown in Figure 4A,B, higher expression of the three stem cell markers was observed in HPAC cells cultured on (PAH/HA)1 and (PAH/HA)3 multilayer nanofilms than in cells cultured on TCPS substrates under both normoxic and hypoxic conditions. In addition, these markers showed higher expression on the surface of the cell aggregates. It is considered that HA-based multilayer film substrate affected the protein localization. Additionally, cells cultured on the TCPS substrate under hypoxic conditions expressed higher levels of CXCR4 and CD44v6 than cells cultured under normoxic conditions. However, CSC marker expression was not increased in cells cultured on multilayer systems under hypoxic conditions. Therefore, colony formation and a hypoxic environment enhance stem cell phenotypes, but an obvious synergistic effect was not observed using this system. CD44 is a widely expressed polymorphic membrane-anchored adhesion molecule that binds HA, and overexpression of CD44 has been shown to be closely associated with the cell growth, the epithelial−mesenchymal transition (EMT), invasion, and

G0/G1, S, and G2 phases after 3 and 7 days of incubation. Figure 3C shows greater percentages of cells in the G0/G1 phase after culture on multilayer nanofilms under hypoxic or normoxic conditions than those after culture on TCPS. In addition, as shown in Figure 3E, the percentages of cells in the G0/G1 phase after culture on multilayer nanofilms for 7 days were substantially increased compared with those found at 3 days of culture. Thus, the formation of colonies/spheres on multilayer nanofilms generated more quiescent cells, particularly after a longer incubation time. It is suggested that this phenomenon is consistent with the previous literature.27 Marker Expression in HPAC Cells Cultured Under Different Conditions. Immunostaining with specific antibodies represents a simple method to analyze the levels of a specific marker. However, a consensus regarding the “best marker(s)” or “single marker” for identifying pancreatic CSCs is currently available. Here, the expression of the stem-cellrelated markers OCT4, CXCR4, and CD44v6 was used to determine the CSC characteristics, as shown in Figure 4A. The quantification of the levels of these markers in cells cultured 38775

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

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Figure 6. Migration and invasion of HPAC cells cultured on HA-based multilayer films and the TCPS substrate under normoxic and hypoxic conditions before and after TGF-β1 induction. (A) Images of HPAC cells incubated under different culture conditions. (B) Quantification of invasion. (C) Percentage of invading cells before and after induction.

tumor progression.29 Furthermore, the expression of CD44 variant isoforms was recently shown to be restricted to subpopulations endowed with the stem cell potential and cancer progression. Among these CD44 variant isoforms, CD44v6 appears to play a major role in tumor progression because of its ability to bind some major cytokines produced by tumor niches.30 Additionally, human pancreatic CSCs expressing the stem cell marker CD133 and the chemokine receptor CXCR4 exhibit higher metastatic activity.31 According to Schimanski et al., CXCR4 expression may correlate with tumor stages, potentially assisting clinicians in predicting the probability of liver metastasis in patients.32 Therefore, we speculated that the heterogeneous island-type colonies that formed on the (PAH/HA)1 substrate and round-type colonies that formed on the (PAH/HA)3 substrate enhance CSC phenotypes. Furthermore, E-cadherin, vimentin, and HIF-1α were also determined as shown in Figure 5A. The results of the quantification of the levels of these markers in cells cultured under normoxic and hypoxic conditions are shown in Figure 5B. HIF-1α expression in cells cultured on both (PAH/HA)1 and (PAH/HA)3 multilayer nanofilms was significantly different from that in cells cultured on the TCPS substrate. In particular, it is revealed that the expression of HIF-1α in the center of colonies is higher than the surface, indicating the hypoxic niche in the center. Previous study has also used the hypoxia probe to visualize and quantify the hypoxic expression of lung cancer spheroid, and it is revealed that spheroids were

highly hypoxic but spheroid-derived peripheral migrating cells were normoxic, relatively.24 The HIF-1α expression results are also consistent with live/dead results, as shown in Figure 3, such that the cells in the center of colonies may be quiescent cells.28 Moreover, colonies cultured on (PAH/HA)1 and (PAH/HA)3 multilayer nanofilms expressed higher levels of Ecadherin, which may be related to colony formation. However, colonies that formed on (PAH/HA)1 and (PAH/HA)3 multilayer nanofilms also expressed higher levels of vimentin than that of cells cultured on TCPS. In the TCPS group, the levels of these three markers were also increased when the cells were cultured in a hypoxic environment when compared with a normoxic environment. The EMT of epithelial cells is defined as the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. Previous study has revealed that a hybrid EMT, an intermediate state between the E and M states, is important in metastatic contexts;33 however, the properties of hybrid types are still unclear, and reliable models are lacking. It is suggested that multilayer nanofilms induced colony formation and regulated the CSC properties, producing cells with hybrid phenotypes. Invasion Assay. An in vitro Matrigel invasion assay was employed to examine the invasion of colonies/spheres cultured on (PAH/HA)1 and (PAH/HA)3 in hypoxic and normoxic environments. As shown in Figure 6A, cells exhibited a low invasion ability after being cultured on both (PAH/HA)1 and (PAH/HA)3 substrates. According to recent studies, CSCs display heterogeneity, and some investigators reported that 38776

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

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ACS Applied Materials & Interfaces

state.34 In the combination system with the hypoxic niche, cells expressed high levels of E-cadherin but lower levels of vimentin, suggesting that this system reduced the number of cells in the hybrid state and resulted in a lower invasion potential. Therefore, we postulated that multilayer nanofilms regulated the cell hybrid metastable state and quiescent CSC properties with a distinct colony phenotype. Drug Resistance Assay: Gemcitabine. An MTT assay was used to evaluate chemotherapeutic drug-induced apoptosis and drug resistance of colonies grown on (PAH/HA)1 and (PAH/HA)3 under normoxic and hypoxic conditions for 3 and 7 days. HPAC cells cultured under different conditions for different incubation times were treated with gradient concentrations of gemcitabine. The relative viability of cells treated with the drug concentration gradient after 3 and 7 days of incubation is shown in Figure 7A,B, respectively. Hypoxiainduced cell stemness leads to drug resistance that has been identified in the previous literature, and CSCs have been identified to be highly correlated with hypoxic conditions.35 The result demonstrated that the exposure to a severe hypoxic niche noticeably enhanced the resistance of all groups to gemcitabine which is consistent with the previous study.35 Previous literature has also revealed that gemcitabine-resistant cells had greater spheroid-forming ability, and one of the ATPbinding cassette (ABC) transporters, ABCB1 (MDR1), was significantly enhanced during the acquisition of gemcitabine resistance.36 In addition, as shown in Figure 7C, cells cultured on (PAH/HA)3 for 7 days exhibited increased drug resistance when compared with cells incubated for only 3 days. However, this phenomenon was not so obvious in cells cultured on the (PAH/HA)1 substrate. Taken together, after 7 days of incubation in a normoxic environment, CSC phenotypes, invasion, and drug resistance were enhanced in cells cultured on multilayer nanofilms. In particular, the compact and dense structure of colonies cultured on (PAH/HA)3 multilayer nanofilms provides a niche to regulate CSC phenotypes. In contrast, the hypoxic niche strongly influenced the drug resistance, and the trend was noticeable in the invasion assay, which revealed that the CSC phenotypes are diverse, and the regulatory factors are complex.

Figure 7. Drug resistance of HPAC cells cultured on HA-based multilayer films and the TCPS substrate under normoxic and hypoxic conditions in the presence of gradient concentrations of gemcitabine for (A) 3 days and (B) 7 days. (C) Comparison of the drug resistance of HPAC cells cultured on HA-based multilayer films. The relative viabilities of cells cultured on different substrates in the presence of various concentrations of gemcitabine were determined using an MTT assay.

some stem cell subsets possess a strong invasive capability, whereas other stem cell subsets are in a quiescent state and do not differentiate.34 The cell cycle assay revealed that a high percentage of cells in colonies/spheres growing on multilayer films were quiescent and expressed high levels of CSC markers. These cells may attain the ability to invade matrix after induction. As expected, a dramatic change was observed in these colonies/spheroids after induction with TGF-β1, and the quantification is shown in Figures 6B and S3. In particular, the greatest increase in the percentage of invading cells was observed in spheroids cultured on the (PAH/HA)3 substrate under normoxic conditions after induction, as shown in Figure 6C. However, the colonies/spheroids in the combination system cultured under hypoxic conditions did not show increased invasion compared with the normoxia groups after induction. As mentioned above, increased CSC marker expression was not observed in cells cultured in the combination system; thus, an obvious synergistic effect was not observed using this system. However, the signaling pathway is still unclear. In particular, the invasion of cells after induction was consistent with the vimentin expression. Multilayer films induced a higher percentage of CSCs to enter quiescent status, particularly general round-type spheroids. Cells in island-type colonies were present in a more heterogeneous population, as shown in the immunostaining results, and the colonies also showed lower invasion potential than that of colonies grown on (PAH/HA)3. Both types of colonies that formed on (PAH/HA)1 and (PAH/HA)3 under normoxic conditions expressed higher levels of E-cadherin and vimentin and displayed a hybrid and intermediate EMT

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CONCLUSIONS This study characterized and classified the regulatory effects of the colony-type and hypoxic niche on CSC properties. The heterogeneous island-type colonies growing on the (PAH/ HA)1 substrate and round-type colonies growing on the (PAH/HA)3 substrate are quiescent and express CSC-related markers, but they show some differences in invasion potential and drug resistance. Multilayer nanofilms may regulate the type of colony that forms, affecting the cell composition and marker expression, and exert a serious impact on invasion. In addition, the hypoxic niche strongly influences the drug resistance. This system represents a useful tool with which to investigate the diverse phenotypes of pancreatic CSCs and to study the regulatory factors.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b14006. 38777

DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779

Research Article

ACS Applied Materials & Interfaces

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Observations of the morphology of single HPAC cells and spheroids after reseeding on TCPS; observations of the morphology of HPAC spheroids subcultured on (PAH/HA)1 and (PAH/HA)3 after 3 days of culture; and quantification of invasive ability of HPAC cells cultured on HA-based multilayer films and the TCPS substrate (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +886-3-2118800 ext. 5985. ORCID

I-Chi Lee: 0000-0001-6527-0077 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Chang Gung Memorial Hospital (Project number: CMRPD1E0361-2) for providing the financial support and Microscopy Center at Chang Gung University for the technical assistance. The authors also acknowledge the grants awarded by the Ministry of Science and Technology in Taiwan (grant number: MOST 105-2221E-182-017). We also gratefully acknowledge Dr. Chia-Ning Shen (Genomics Research Center, Academia Sinica) for providing the cell line.

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DOI: 10.1021/acsami.8b14006 ACS Appl. Mater. Interfaces 2018, 10, 38769−38779